The serine proteinase inhibitory proteins of the human intervertebral disc: Their isolation, characterization and variation with ageing and degeneration

Serine proteinase inhibitory proteins (SPIs) were extracted from human disc tissues using 2 M GuHCl and subjected to CsCl density gradient ultracentrifugation. The SPIs recovered in the low buoyant density fractions (ϱ ≤ 1.35 g/ml) were purified by a combination of gel-permeation, ion-exchange, trypsin affinity, and reverse-phase high performance chromatographies. Characterisation of the major disc SPI by polyacrylamide gel electrophoresis, isoelectric focussing, enzyme inhibition and pH stability studies indicated that this small molecular weight (12–14 kDa), highly basic (pI > 9.5), acid-stable but alkaline-labile protein possessed potent inhibitory activity against bovine pancreatic trypsin and chymotrypsin, and human leukocyte elastase and cathepsin G. Two-major and two-minor low molecular weight cationic SPI species were identified by reverse-phase HPLC. The predominant species was identical to a human articular cartilage SPI sharing amino terminal sequence homology with the mucus proteinase inhibitors (MPIs). It also cross-reacted with an antiserum to the MPIs and behaved identically to secretory leucocyte proteinase inhibitor (SLPI) when examined by reverse phase HPLC, and SDS PAGE. A higher molecular weight (54 kDa), anionic (pI ∼ 4.6) SPI was also purified and identified as α₁-proteinase inhibitor (α₁-PI). Quantification of α₁-PI and the small molecular weight cationic disc inhibitors indicated that the latter were depleted in morphologically degenerate disc tissues while levels of α₁-PI were somewhat higher although a large proportion of the α₁-PI was inactive. A depletion of total SPI levels was evident overall in degenerate discs suggesting a functional role for these inhibitory proteins in the maintenance of IVD matrix homeostasis.